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目的:在大肠杆菌中表达具有生物学活性的白细胞介素-33(mIL-33)。方法:用PCR技术扩增小鼠IL-33成熟蛋白的编码区,与原核表达载体pET-44连接,构建pET-44-mIL-33表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达,镍柱亲和层析法纯化目的蛋白,SDS-PAGE分析表达产物,MTT法测定纯化IL-33的生物学活性。结果:成功构建了pET-44-mIL-33原核表达载体,并在大肠杆菌中表达出可溶性mIL-33,SDS-PAGE分析纯化产物纯度为95%,表达产量为95mg/L。细胞增殖实验表明IL-33有抑制P815细胞增殖的活性。结论:获得了有活性的重组IL-33蛋白纯品,为后续功能研究奠定了基础。
OBJECTIVE: To express the biologically active interleukin-33 (mIL-33) in Escherichia coli. Methods: The coding region of mature mouse IL-33 protein was amplified by PCR and ligated with prokaryotic expression vector pET-44 to construct pET-44-mIL-33 expression vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) Purification of the target protein by nickel column affinity chromatography, SDS-PAGE analysis of expression products, the biological activity of purified IL-33 was measured by MTT method. Results: The prokaryotic expression vector pET-44-mIL-33 was successfully constructed and soluble mIL-33 was expressed in E. coli. The purity of purified product was 95% by SDS-PAGE and the yield was 95 mg / L. Cell proliferation experiments showed that IL-33 can inhibit the proliferation of P815 cells. Conclusion: Obtained active recombinant recombinant IL-33 protein, which laid the foundation for the follow-up study of function.